We are pursuing a replication-competent Adenovirus (Ad)-recombinant priming/subunit boosting approach as a prophylactic vaccine for HIV/AIDS. Studies showing that the vaccine regimen elicits humoral, cellular, and mucosal immunity together with significant protection in non-human primate models have moved this approach towards Phase I human trials. At the same time, further pre-clinical studies are evaluating the utility of recombinants encoding additional viral genes, investigating new combination regimens, and exploring routes of immunization for optimal elicitation of immune responses and protective efficacy. Previously, we reported that priming with multigenic Ad-SIV recombinants followed by boosting with either gp120 or a peptide polymer representing the CD4 binding site of the envelope protein elicited potent protection in 39% of immunized macaques. In recent follow-up studies we have demonstrated durability of the protection, as 73% of the protected macaques resisted a second SIV challenge one year following the first exposure with no additional immunizations. This durable protection was associated with SIV-specific CD8+ T cells. However, we have also shown that the Ad-recombinant prime/protein boost regimen elicits high titered anti-envelope antibodies, including those that mediate antibody-dependent cellular cytotoxicity (ADCC). Vaccine-induced ADCC activity correlated with reduced acute phase viremia. Subsequently, we demonstrated that an Ad-HIV recombinant prime/envelope protein boost regimen induced high titer antibodies that mediated broad ADCC activity across HIV clades, a significant finding for future vaccines that must protect against the global spectrum of HIV strains. Investigation of additional combination vaccine regimens have shown that administration of Ad-HIV recombinants encoding HIVenv and HIVtat, followed by boosting with HIV Env and Tat proteins elicits enhanced protective efficacy against a SHIV89.6P challenge in comparison to regimens based on Tat alone or multigenic approaches. The enhanced protection was associated with antibody responses to both Tat and Env. Mechanisms associated with this enhanced protection are under investigation. As mucosal immunity is critical for an HIV vaccine, more recently we have explored mucosal routes of immunization and have shown that sequential intranasal/oral administration of Ad-SIV recombinants is more immunogenic than a regimen based on two sequential oral administrations. The former regimen has also resulted in greater reduction of acute viremia following a mucosal SIV challenge. On-gong studies are investigating the value of adding DNA vaccine encoding cytokines into our vaccine strategy and evaluating novel envelope subunit vaccines as booster immunizations. Importantly, these pre-clinical studies contribute to identification of immune correlates of protection, and development of new combination strategies that induce synergistic immunity and protective efficacy. This new knowledge will inform future vaccine design and define vaccine regimens that will merit testing in clinical trials.